This invention is generally in the area of antibodies to plasma proteins, specifically Protein C, and methods for use thereof.
Protein C is a vitamin K-dependent plasma protein zymogen to a serine protease. Upon activation it becomes a potent anticoagulant. Activated protein C acts through the specific proteolysis of the procoagulant cofactors, factor VIIIa and factor Va. This activity requires the presence of another vitamin K-dependent protein, protein S, calcium and a phospholipid (presumably cellular) surface. As described in Hemostasis and Thrombosis: Basic Principles and Clinical Practice 2nd Ed., Colman, R. W., et al., p. 263 (J.B.Lippincott, Philadelphia, Pa. 1987), protein C circulates in a two-chain form, with the larger, heavy chain bound to the smaller light chain through a single disulfide link. A small proportion of the protein also circulates in a single chain form, where a Lys-Arg dipeptide in the molecule connects the light chain directly to the heavy chain.
Protein C is activated to activated protein C (APC). Thrombin is capable of activating protein C by the specific cleavage of the Arg12-Leu13 bond in the heavy chain. In vivo, in the presence of physiological concentrations of calcium, the rate of this activation is enhanced dramatically when thrombin is bound to the endothelial cell cofactor, thrombomodulin. Matschiner, et al., Current Advances in Vitamin K Research, pp. 135–140, John W. Suttie, ed. (Elsevier Science Publishing Co., Inc. 1988) have further reviewed the role of the Vitamin K dependent proteins in coagulation.
Protein C has been shown to have major importance in vivo. Patients deficient in protein C, or its cofactor, protein S, show pronounced thrombotic tendencies. Babies born totally deficient in protein C exhibit massive disseminated intravascular coagulation (DIC) and a necrotic syndrome which leads to death within the first few weeks of life if untreated. Activated protein C has also been shown to protect animals against the coagulopathic and lethal effects of endotoxin shock, as described by Taylor, et al., in J. Clin. Invest. 79, 918–925 (1987).
As first reported by Kisiel, in J. Clin. Invest. 64, 761–769 (1979), Protein C was originally isolated in semi-pure form from plasma using classic protein purification techniques, including barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. This procedure was vastly improved and facilitated by the discovery of a unique antibody to Protein C, designated HPC-4, described by Stearns, et al., in J. Biol. Chem. 263(2), 826–832 (1988). As detailed by Esmon, et al., at the Joint IABS/CSL Symposium on Standardization in Blood Fractionation including Coagulation Factors, Melbourne, Australia 1986 (reported in Develop. Biol. Standard., 67, 51–57 (S. Karger, Basel, 1987), Protein C can be isolated from human plasma by batch adsorption of diluted heparinized plasma on QAE Sephadex, washing with buffered 0.15 M NaCl and eluting with 0.5 M NaCl, recalcifying and batch adsorbing with HPC-4, then washing with a Ca2+ containing buffer and eluting the Protein C with an EDTA containing buffer. HPC-4 is a calcium-dependent monoclonal antibody to human protein C. The epitope recognized by the antibody has been identified and corresponds to the stretch of amino acids in the zymogen of protein C which spans the thrombin cleavage site. Activated protein C is not recognized by HPC-4. HPC-4 is disclosed and claimed in U.S. Pat. No. 5,202,253 to Esmon, et al.
Several antibodies to human protein C have been reported, for example, by Laurell, et al., FEBS Letts. 191(1), 75–81 (1985); Wakabayashi, et al., J. Biol. Chem. 261, 11097–11105 (1986); Sugo, et al., Thromb. Hemost. Abstrs., Brussells, 229 (1987); and Ohlin, et al., J. Biol. Chem. 262, 13798–13804 (1988). Some of these are calcium dependent, for example, one of the antibodies reported by Laurell, et al. However, as far as can be determined in the published reports, this dependence is due to the requirement for calcium binding to the light chain of protein C and the antibodies recognize epitopes on the light chain. Other antibodies recognize the region around the thrombin cleavage site on the heavy chain, but these are not calcium dependent. The HPC-4 antibody of Ohlin, et al., is Ca2+ dependent but is not directed against the activation region, and is therefore different from the antibody described in Stearns, et al., and in U.S. Pat. No. 5,202,253 to Esmon, et al.
All of the other antibodies that bind to the Ca2+ stabilized regions of Protein C recognize both Protein C and the activated form of Protein C. Situations may arise in which the protein uncontaminated by its active form is desirable. This is particularly the case with reference to therapeutic uses of the antibody to inhibit Protein C activation.
Blockage of the natural anticoagulant pathways, in particular the protein C pathway, uses the natural procoagulant properties of the tumor to target the tumor capillaries for microvascular thrombosis, leading to hemorrhagic necrosis of the tumor, as described in U.S. Pat. No. 5,147,638 to Esmon, et al. HPC-4 is a preferred antibody for use in this method for the treatment of solid tumors, either alone or in conjunction with biological response modifiers, chemotherapy or radiation treatments.
Tumors contain proteins which predispose to the formation of blood clots in the vessels in the tumor bed. Tumors also contain other proteins and cellular elements which prevent thrombosis of tumor blood vessels. Tumor necrosis results from altering the hemostatic balance between procoagulant and anticoagulant mechanisms to favor thrombosis of the tumor microvasculature. The hemostatic balance of the tumor can be altered by blocking the conversion of protein C to its active form (activated protein C). The procoagulant mechanisms present in the tumor bed will then function without opposition and cause thrombosis of the tumor vessels. The epitope for the HPC-4 antibody spans the activation site in protein C and as a result blocks protein C activation. As an experimental tool it is important to note that the antibody cross-reacts with protein C from canine, porcine and at least two primate plasmas, baboon and marmoset. It does not cross-react with bovine or mouse protein C. The inhibitory effect can be reversed instantly by administration of activated protein C to which the antibody does not bind. The antibody therefore provides a means to selectively inhibit the protein C pathway in vivo and to reverse the process if thrombotic complications ensue at sites other than the tumor. The Protein C blocking agent is preferably administered in combination with a cytokine that stimulates natural killer and lymphokine-activated killer cell-mediated cytotoxicity, activates macrophages, stimulates Fc receptor expression on mononuclear cells and antibody-dependent cellular cytotoxicity, enhances HLA class II antigen expression, and/or stimulates procoagulant activity, such as tumor necrosis factor (TNF), interleukin-1 (IL-1), interleukin-2 (IL-2), gamma interferon (gamma-IFN), or granulocyte-macrophage colony stimulating factor (GMCSF). Alternatively, an agent such as endotoxin, or the purified liposaccharide (LPS) from a gram negative bacteria such as E. coli, can be used to elicit production of cytokines such as TNF.
HPC-4, despite its wonderful properties, is a murine antibody. It would be advantageous to be able to provide a humanized form of the antibody which is non-immunogenic or less immunogenic. In order to construct a humanized form of HPC-4 it is essential to know the sequence of the hypervariable regions of this antibody. Then using conventional mutagenesis methods developed in molecular biology it is possible to replace the sequence of hypervariable regions of an unrelated human antibody with the sequences of HPC-4 hypervariable regions. Such an approach has been successfully used in the humanization of other antibodies. Furthermore by knowing the sequence of the hypervariable region it may be possible to synthesize short peptides corresponding to the hypervariable regions of the HPC-4 antibody which could mimic HPC-4 and bind to the same region on protein C and prevent activation of protein C by thrombin-thrombomodulin complex. Such peptides could be very effective in disease states where promoting of the clotting is desired.
It is therefore an object of the present invention to provide a recombinant Ca2+ dependent antibody which binds to the activation region of Protein C like HPC-4.
It is a further object of the present invention to provide a DNA sequence encoding the hypervariable region of an antibody like HPC-4.
It is a still further object of the present invention to provide a method and means for using this Ca2+ dependent antibody for therapeutic purposes.
It is yet another object of the present invention to provide this Ca2+ dependent antibody, antibodies, peptide derivatives and conjugates thereof, for diagnostic purposes.